Plasma membrane expression of the neuronal glutamate transporter EAAC1 is regulated by glial factors: evidence for different regulatory pathways associated with neuronal maturation

At the glutamatergic synapse the neurotransmitter is removed from the synaptic cleft by high affinity amino acid transporters located on neurons (EAAC1) and astrocytes (GLAST and GLT1), and a coordinated action of these cells is necessary in order to regulate glutamate extracellular concentration. We show here that treatment of neuronal cultures with glial soluble factors (GCM) is associated with a redistribution of EAAC1 and GLAST to the cell membrane and we analysed the effect of membrane cholesterol depletion on this regulation.

In enriched neuronal culture (90% neurons and 10% astrocytes), GCM treatment for 10 days increases EAAC1 and GLAST cell surface expression with no change in total expression. In opposite, GLT1 surface expression is not modified by GCM but total expression is increased. When cholesterol is acutely depleted from the membrane by 10 mM methyl-beta-cyclodextrin (β5-MCD, 30 min), glutamate transport activity and cell surface expressions of EAAC1 and GLAST are decreased in the enriched neuronal culture treated by GCM. In pure neuronal culture addition of GCM also increases EAAC1 cell membrane expression but surprisingly acute treatment with β5-MCD decreases glutamate uptake activity but not EAAC1 cell membrane expression. By immunocytochemistry a modification in the distribution of EAAC1 within neurons was undetectable whatever the treatment but we show that EAAC1 was no more co localized with Thy-1 in the enriched neuronal culture treated by GCM suggesting that GCM have stimulated polarity formation in neurons, an index of maturation.

In conclusion we suggest that different regulatory mechanisms are involved after GCM treatment, glutamate transporter trafficking to and from the plasma membrane in enriched neuronal culture and modulation of EAAC1 intrinsic activity and/or association with regulatory proteins at the cell membrane in the pure neuronal culture. These different regulatory pathways of EAAC1 are associated with different neuronal maturation stages.     

Platelet-derived growth factor rapidly increases activity and cell surface expression of the EAAC1 subtype of glutamate transporter through activation of phosphatidylinositol 3-kinase

Na(+)-dependent glutamate transporters are the primary mechanism for removal of excitatory amino acids (EAAs) from the extracellular space of the central nervous system and influence both physiologic and pathologic effects of these compounds. Recent evidence suggests that the activity and cell surface expression of a neuronal subtype of glutamate transporter, EAAC1, are rapidly increased by direct activation of protein kinase C and are decreased by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). We hypothesized that this regulation could be analogous to insulin-induced stimulation of the GLUT4 subtype of glucose transporter, which is dependent upon activation of PI3-K. Using C6 glioma, a cell line that endogenously and selectively expresses EAAC1, we report that platelet-derived growth factor (PDGF) increased Na(+)-dependent L-[(3)H]-glutamate transport activity within 30 min. This effect of PDGF was not due to a change in total cellular EAAC1 immunoreactivity but was instead correlated with an increase cell surface expression of EAAC1, as measured using a membrane impermeant biotinylation reagent combined with Western blotting. A decrease in nonbiotinylated intracellular EAAC1 was also observed. These studies suggest that PDGF causes a redistribution of EAAC1 from an intracellular compartment to the cell surface. These effects of PDGF were accompanied by a 35-fold increase in PI3-K activity and were blocked by the PI3-K inhibitors, wortmannin and LY 294002, but not by an inhibitor of protein kinase C. Other growth factors, including insulin, nerve growth factor, and epidermal growth factor had no effect on glutamate transport nor did they increase PI3-K activity. These studies suggest that, as is observed for insulin-mediated translocation of GLUT4, EAAC1 cell surface expression can be rapidly increased by PDGF through activation of PI3-K. It is possible that this PDGF-mediated increase in EAAC1 activity may contribute to the previously demonstrated neuroprotective effects of PDGF.     

Insulin increases glutamate transporter GLT1 in cultured astrocytes

The astroglial cell-specific glutamate transporter subtype 2 (excitatory amino acid transporter 2, GLT1) plays an important role in excitotoxicity that develops after damage to the central nervous system (CNS) is incurred. Both the protein kinase C signaling pathway and the epidermal growth factor (EGF) pathway have been suggested to participate in the modulation of GLT1, but the modulatory mechanisms of GLT1 expression are not fully understood. In the present study, we aimed to evaluate the effects of insulin on GLT1 expression. We found that short-term stimulation of insulin led to the upregulation of both total and surface expressions of GLT1. Akt phosphorylation increased after insulin treatment, and triciribine, the inhibitor of Akt phosphorylation, significantly inhibited the effects of insulin. We also found that the upregulation of GLT1 expression correlated with increased kappa B motif-binding phosphoprotein (KBBP) and GLT1 mRNA levels. Our results suggest that insulin may modulate the expression of astrocytic GLT1, which might play a role in reactive astrocytes after CNS injuries.     

Dopamine denervation of the prefrontal cortex increases expression of the astrocytic glutamate transporter GLT‐1

Both dopamine and glutamate are critically involved in cognitive processes such as working memory. Astrocytes, which express dopamine receptors, are essential elements in the termination of glutamatergic signaling: the astrocytic glutamate transporter GLT‐1 is responsible for > 90% of cortical glutamate uptake. The effect of dopamine depletion on glutamate transporters in the prefrontal cortex (PFC) remains unknown. In an effort to determine if astrocytes are a locus of cortical dopamine–glutamate interactions, we examined the effects of chronic dopamine denervation on PFC protein and mRNA levels of glutamate transporters. PFC dopamine denervation elicited a marked increase in GLT‐1 protein levels, but had no effect on levels of other glutamate transporters; high‐affinity glutamate transport was positively correlated with the extent of dopamine depletion. GLT‐1 gene expression was not altered. Our data suggest that dopamine depletion may lead to post‐translational modifications that result in increased expression and activity of GLT‐1 in PFC astrocytes.

     

Differential expression of the glutamate transporter GLT-1 in pancreas

The glutamate uptake transporter GLT-1 is best understood for its critical role in preventing brain seizures. Increasing evidence argues that GLT-1 also modulates, and is modulated by, metabolic processes that influence glucose homeostasis. To investigate further the potential role of GLT-1 in these regards, the authors examined GLT-1 expression in pancreas and found that mature multimeric GLT-1 protein is stably expressed in the pancreas of wild-type, but not GLT-1 knockout, mice. There are three primary functional carboxyl-terminus GLT-1 splice variants, called GLT-1a, b, and c. Brain and liver express all three variants; however, the pancreas expresses GLT-1a and GLT-1b but not GLT-1c. Quantitative real time-PCR further revealed that while GLT-1a is the predominant GLT-1 splice variant in brain and liver, GLT-1b is the most abundant splice variant expressed in pancreas. Confocal microscopy and immunohistochemistry showed that GLT-1a and GLT-1b are expressed in both islet β- and α-cells. GLT-1b was also expressed in exocrine ductal domains. Finally, glutamine synthetase was coexpressed with GLT-1 in islets, which suggests that, as with liver and brain, one possible role of GLT-1 in the pancreas is to support glutamine synthesis.     

Glial soluble factors regulate the activity and expression of the neuronal glutamate transporter EAAC1: implication of cholesterol

A co-ordinated regulation between neurons and astrocytes is essential for the control of extracellular glutamate concentration. Here, we have investigated the influence of astrocytes and glia-derived cholesterol on the regulation of glutamate transport in primary neuronal cultures from rat embryonic cortices. Glutamate uptake rate and expression of the neuronal glutamate transporter EAAC1 were low when neurons were grown without astrocytes and neurons were unable to clear extracellular glutamate. Treatment of the neuronal cultures with glial conditioned medium (GCM) increased glutamate uptake Vmax, EAAC1 expression and restored the capacity of neurons to eliminate extracellular glutamate. Thus, astrocytes up-regulate the activity and expression of EAAC1 in neurons. We further showed that cholesterol, present in GCM, increased glutamate uptake activity when added directly to neurons and had no effect on glutamate transporter expression. Furthermore, part of the GCM-induced effect on glutamate transport activity was lost when cholesterol was removed from GCM (low cholesterol-GCM) and was restored when cholesterol was added to low cholesterol-GCM. This demonstrates that glia-derived cholesterol regulates glutamate transport activity. With these experiments, we provide new evidences for neuronal glutamate transport regulation by astrocytes and identified cholesterol as one of the factors implicated in this regulation.     

Effect of benzodiazepines on the epithelial and neuronal high-affinity glutamate transporter EAAC1

EAAC1-mediated glutamate transport concentrates glutamate across plasma membranes of brain neurons and epithelia. In brain, EAAC1 provides a presynaptic uptake mechanism to terminate the excitatory action of released glutamate and to keep its extracellular concentration below toxic levels. Here we report the effect of well known anxiolytic compounds, benzodiazepines, on glutamate transport in EAAC1-stably transfected Chinese hamster ovary (CHO) cells and in EAAC1-expressing Xenopus laevis oocytes. Functional properties of EAAC1 agreed well with already reported characteristics of the neuronal high-affinity glutamate transporter (Km D-Asp,CHO cells: 2.23+/-0.15 microM; Km D-Asp,oocytes: 17.01+/-3.42 microM). In both expression systems, low drug concentrations (10-100 microM) activated substrate uptake (up to 200% of control), whereas concentrations in the millimolar range inhibited (up to 50%). Furthermore, the activation was more pronounced at low substrate concentrations (1 microM), and the inhibition was attenuated. The activity of other sodium cotransporters such as the sodium/D-glucose cotransporter SGLT1, stably transfected in CHO cells, was not affected by benzodiazepines. In electrophysiological studies, these drugs also failed to change the membrane potential of EAAC1-expressing Xenopus laevis oocytes. These results suggest a direct action on the glutamate transporter itself without modifying the general driving forces. Thus, in vivo low concentrations of benzodiazepines may reduce synaptic glutamate concentrations by increased uptake, providing an additional mechanism to modulate neuronal excitability.     

Arginine 445 controls the coupling between glutamate and cations in the neuronal transporter EAAC-1

The substrate-binding sites in membrane transporters are alternately accessible from either side of the membrane, but the molecular basis of how this alternate opening of internal and external gates is achieved is largely unknown. Here we present data indicating that, in the neuronal electrogenic sodium- and potassium-coupled glutamate transporter EAAC-1, the substrate-binding site and one of the gates, or a residue controlling the gating process, are in close physical proximity. Arginine 445, located only two residues away from a residue implicated in glutamate binding (Bendahan, A., Armon, A., Madani, N., Kavanaugh, M. P., and Kanner, B. I. (2000) J. Biol. Chem. 275, 37436-37442), has been mutated to serine (R445S). Upon expression in oocytes, measurements of l-[(3)H]-glutamate transport under voltage clamp reveal that the charge/flux ratio for l-glutamate at -60 mV is approximately 30-fold higher than that of the wild type. Also, with d-aspartate, R445S exhibits an approximately 15-fold increase in this ratio. In contrast to the wild type, the reversal potential of the substrate-dependent currents in R445S shifts to more negative potentials when either the external sodium or potassium concentration is decreased. These findings indicate that these two cations are the main current carriers in the R445S mutant. Introduction of a methionine or a glutamine, but not a lysine, at position 445 gives rise to a phenotype similar to R445S. Therefore, it seems that the elimination of a positive charge in the vicinity of the substrate-binding site converts the transporter into a glutamate-gated cation-conducting pathway.